The use of rodent, especially mouse, monoclonal antibodies for therapeutic and in vivo diagnostic applications in man was found to be limited by immune responses made by patients to the rodent antibody. The development of so-called “HAMA” (human anti-mouse antibody) responses in patients was shown to limit the ability of antibodies to reach their antigenic targets resulting in a reduced effectiveness of the antibodies. In order to reduce the HAMA response, chimeric antibodies were developed (see, for example, WO-A-8909622) in which the mouse variable (V) regions were joined to human constant (C) regions. Such antibodies have proved clinically useful although the mouse V region component still provides the basis for generating immunogenicity in patients (see, for example, LoBuglio et al., Proc. Nat'l. Acad. Sci. USA, 86: 4220-4224 (1989)). Therefore, technology for humanized antibodies were developed whereby the complementarity determining regions or “CDRs” from the rodent antibody were transplanted onto human V regions and joined to human C regions to create humanized antibodies whereby the only non-human components were the CDRs which were adjacent to human V region “frameworks”. The transplanted CDRs corresponded either to hypervariable regions as defined by Kabat et al. (“Sequences of Proteins of Immunological Interest”, Kabat E., et al., U.S. Dept. of Health and Human Services, 1983) or to the hypervariable loops in 3-dimensional structures of antibodies (Chothia and Lesk, J Mol. Biol., 196: 901-917 (1987)). One of the first examples of such humanized antibodies by Riechmann et al. (Nature, 332: 323-326 (1988)) illustrated, however, that simple transplantation of CDRs often resulted in reduced affinity of the humanized antibody and consequently that the introduction of certain non-human amino acids (i.e. from the corresponding position in the rodent sequence) in the human V region framework as required in order to restore affinity. A number of methods have been proposed for the substitution of human framework residues in order to restore affinity including those disclosed in EP-A-0239400, EP-A-0438310, WO-A-9109967 and WO-A-9007861. In particular, patent publications by Protein Design Labs., Inc. (e.g. WO-A-9007861 and related EP-B-0451216) purport to provide a general method for producing humanized antibodies in which one or more human framework residues are altered in order to restore binding affinity.
A common aspect of all of the above mentioned methods for production of chimeric or humanized antibodies is that the objective of these methods was to create antibodies which are substantially non-immunogenic in humans (e.g. EP-B-0451216, page 3, line 6). However, the means for achieving this objective has been the introduction into the rodent antibody of as much human sequence as possible and it has been assumed that such a general introduction of human sequence will render the antibodies non-immunogenic. It is known that certain short peptide sequences (“epitopes”) can be immunogenic in humans and none of the methods for chimeric or humanized antibodies have considered how to eliminate or avoid such epitopes in the resultant antibody. Furthermore, most of the methods (e.g. EP-B-0451216) have advocated the introduction of non-human amino acids into human V region frameworks without considering the possible creation of immunogenic epitopes, and none of the methods has provided any means for avoiding or eliminating immunogenic epitopes at framework:CDR junctions and, where practical, within CDRs themselves. Thus, of the methods devised with the objective of creating substantially non-immunogenic antibodies, none can be considered as actually achieving the creation of such substantially non-immunogenic antibodies. The same can be said of proteins (especially therapeutic proteins) other than antibodies.